Three-dimensional cell to tissue development process

ABSTRACT

The present invention relates to an improved three-dimensional cell to tissue development process using a specific time varying electromagnetic force, pulsed, square wave, with minimum fluid shear stress, freedom for 3-dimensional spatial orientation of the suspended particles and localization of particles with differing or similar sedimentation properties in a similar spatial region.

ORIGIN OF THE INVENTION

The invention described herein was made in part by an employee of theUnited States Government and may be manufactured and used by and for theGovernment of the United States for governmental purposes without thepayment of any royalties thereon of therefor.

FIELD OF THE INVENTION

The present invention relates to a 3-dimensional cell to tissue andmaintenance process, more particularly to methods of culturing cells ina culture environment, either in space or in a gravity field, withminimum fluid shear stress, freedom for 3-dimensional spatialorientation of the suspended particles and localization of particleswith differing or similar sedimentation properties in a similar spatialregion. The process is a dramatic improvement over prior processes inthat the production rate and amount is significantly increased byapplying a time varying electromagnetic force of from 0.05 gauss to 0.5gauss to the culture environment during the culturing of the cells.

BACKGROUND OF THE INVENTION

Cell culture processes have been developed for the growth of single cellbacteria, yeast and molds which are resistant to environmental stressesor are encased with a tough cell wall. Mammalian cell culture, however,is much more complex because such cells are more delicate and have morecomplex nutrient and other environmental requirements in order tomaintain viability and cell growth. Large-scale cultures of bacterialtype cells are highly developed and such culture processes are lessdemanding and are not as difficult to cultivate as mammalian cells.These techniques are highly empirical and a firm theoretical basis isnot developed. Bacterial cells can be grown in large volumes of liquidmedium and can be vigorously agitated without any significant damage.Mammalian cells, on the other hand, cannot withstand excessive turbulentaction without damage to the cells and must be provided with a complexnutrient medium to support growth.

In addition, mammalian cells have other special requirements; inparticular most animal cells must attach themselves to some substratesurface to remain viable and to duplicate. On a small scale, mammaliancells have been grown in containers with small microwells to providesurface anchors for the cells. However, cell culture processes formammalian cells in such microwell containers generally do not providesufficient surface area to grow mammalian cells on a sufficiently largescale basis for many commercial or research applications. To providegreater surface areas, microcarrier beads have been developed forproviding increased surface areas for the cultured cells to attach.Microcarrier beads with attached cultured cells require agitation in aconventional bioreactor vessel to provide suspension of the cells,distribution of fresh nutrients, and removal of metabolic wasteproducts. To obtain agitation, such bioreactor vessels have usedinternal propellers or movable mechanical agitation devices which aremotor driven so that the moving parts within a vessel cause agitation inthe fluid medium for the suspension of the microcarrier beads andattached cells. Agitation of mammalian cells, however, subjects them tohigh degrees of shear stress that can damage the cells and limit orderedassembly of these cells according to cell derived energy. These shearstresses arise when the fluid media has significant relative motion withrespect to vessel walls, impellers, or other vessel components. Cellsmay also be damaged in bioreactor vessels with internal moving parts ifthe cells or beads with cells attached collide with one another orvessel components.

In addition to the drawbacks of cell damage, bioreactors and othermethods of culturing mammalian cells are also very limited in theirability to provide conditions that allow cells to assemble into tissuesthat simulate the spatial 3-dimensional form of actual tissues in theintact organism. Conventional tissue culture processes limit, forsimilar reasons, the capacity for cultured tissues to express a highlyfunctionally specialized or differentiated state considered crucial formammalian cell differentiation and secretion of specialized biologicallyactive molecules of research and pharmaceutical interest. Unlikemicroorganisms, the cells of higher organisms such as mammals formthemselves into high order multicellular tissues. Although the exactmechanisms of this self-assembly are not known, in the cases that havebeen studied thus far, development of cells into tissues has been foundto be dependent on orientation of the cells with respect to each other(the same or different type of cell) or other anchorage substrate and/orthe presence or absence of certain substances (factors) such ashormones, autocrines, or paracrines. In summary, no conventional cultureprocess is capable of simultaneously achieving sufficiently low shearstress, sufficient 3-dimensional spatial freedom, and sufficiently longperiods for critical cell interactions (with each other or substrates)to allow excellent modeling of in vivo tissue structure.

U.S. Pat. No. 5,155,035, Wolf et al., provides a method that overcomesprior problems without subjecting the cells to destructive amounts ofshear, but the production rate of such process is insufficiently low tobe of substantial commercial value. The current invention improves onthat process by subjecting the culture medium to a time varyingelectromagnetic force to increase the production rate to commerciallysignificant levels.

A paper entitled: “The Clinostat—A Tool For Analyzing The Influence ofAcceleration On Solid-Liquid Systems” by W. Briegleb, published by theproceedings of a workshop on Space biology, Cologne Germany, on Mar. 11,1983, (ESASP-206, May 1983). In this paper, clinostat principles aredescribed and analyzed relative to gravity affects. Some clinostatexperiments are described including experiments where monocellularsuspended organisms (protozoans) are placed within cylinders which arerotated about a horizontal axis.

A paper entitled, “The Large-Scale Cultivation of Mammalian Cells”, byJoseph Feder and William R. Tolbert, published in the ScientificAmerican, January 1983, Vol. 248, No. 1. Pgs. 36-43. In this paper,agitation of the cells is described as required to keep the cellssuspended in the medium and describes a turbine agitator, a marinepropeller agitator, and a vibrating mixer for mixing. The paper alsodescribes a perfusion reactor in which four slowly rotating flexiblesheets of monofilament nylon provide agitation The sheets are rotatedabout a vertical axis while the medium in the main vessel iscontinuously pumped to the satellite filter vessel. The filter retainsthe cells which are pumped along with the remainder medium back into thevessel for further proliferation. A paper entitled, “Large Scale CellCulture Technology”, William R. Tolbert, Joseph Feder, Monsanto Company,St. Louis, Mo., Annual Reports on Fermentation Processes, Vol. 6, 1983,discloses a flat plate hollow fiber system of high culture surface areadescribed as allowing culture of anchorage dependent cells andaccumulation of cell secretor products. A culture vessel with flexiblespiral vanes for suspension of microcarriers and a fluidized bed withmedia perfusion is described.

A paper entitled, “Gravisensitivity of the Acellular, Slime, Mold,Physarum, Polycephalum Demonstrated on the Fast Rotating Clinostat”, byIngrid Block and Wolfgang Brigley, published in the European Journal ofCell Biology 41, Pgs. 44-50, 1986. This paper described rotation of aculture vessel about a horizontal axis for the simulation ofweightlessness.

A paper entitled, “Cell and Environment Interactions in TumorMicroregions: The Multicell Spheroid Model”; by Robert M. Sutherland,Science 240: 177-184; (1988) discloses the use of multicell spheroids,without attachment substrates, of tumor cells to study cell andenvironment interactions in tumors. Conventional culture, processes areutilized to produce limited size and viable tumor cell aggregates.

Cell cultures from various bio-reactors, including a prototype of a slowturning lateral vessel (STLV) designed for batch culturing of cells werepresented at a poster session at the First Canadian Workshop on R & DOpportunities on Board the Space Station, National Research CouncilCanada, May 6-8, 1987, Ottawa, Canada, and published in the Proceedings“Spacebound '87”, as a paper entitled “Growth and Maintenance ofAnchorage Dependent Cells in Zero Headspace Bioreactor Systems DesignedFor Microgravity”, by Lewis et al.

A paper entitled, “Physical Mechanisms of Cell Damage in MicrocarrierCell Culture Bioreactors”, Robert S. Cherry and Eleftherios TerryPoportsakis, Biotechnology and Bioengineering, Vol. 32, Pp. 1001-1014(1988) discloses mechanisms for damage of anchorage dependent cellscultured on microcarrier beads. Bridging of cells across beads to formbead aggregates is described as a damage mechanism due to breaking ofthese bridges from mechanical mechanisms.

SUMMARY OF THE INVENTION

The process of the present invention provides methods for enhancing theculture of cells and living 3-dimensional tissues by controlling thefluid mechanical environment to achieve the desired culturecharacteristics. More specifically, the process of the present inventionallows enhanced capability to simultaneously achieve a cultureenvironment with reduced fluid shear stress, freedom for 3-dimensionalspatial orientation (of suspended particles), and localization ofparticles with differing (or similar) sedimentation properties in asimilar spatial region (collocation). This enhanced culture processallows orderly assembly of cells and attachment substrates into higherorder tissue-like structures. Carrying out the culture process of thepresent invention in reduced gravity, as in space, allows an immenseexpansion of these operating limitations. However even in unit gravitythe process of this invention allows a large improvement overconventional culture processes for typical tissues of interest. Theminimal fluid shear stress obtainable in unit gravity is determined bythe gravitationally induced motion of the suspended particles throughthe culture medium and is far less in the process of the presentinvention than that obtainable by culture processes heretofore used. Ofcritical importance to this invention is subjecting the culture to atime varying electromagnetic force (pulsed, square wave) of from 0.05 to0.5 gauss during the culture process.

A 3-dimensional culture environment which initially is controlled tohave essentially no fluid shear stress which is part of the process ofthe present invention allows higher densities of healthy living cellsand allows the maintenance or growth of cells into higher order3-dimensional multicellular tissue-like structures with larger size,improved viability, and finer delicate structure than other culturingprocesses. These cultured tissues are higher fidelity models of theactual in vivo tissue and are therefore important for research andclinical applications. In addition delicate cell types may be culturedwith improved viability with the processes of the present invention. Inthe process of the present invention, higher levels of cell and tissuedifferentiation may be obtained by allowing freedom for the spatialorientation of cells with respect to each other, exchange of solublechemical signals, intercellular mechanical signals, cellular toinsoluble substrate interactions, and expression of the tissuedifferentiation state resulting from these inductive mechanisms.Subjecting the cells to the specified time varying electromagnetic forcedramatically increases the cell growth and differentiation to an extentthat the cell production becomes commercially important.

The minimal shear stress level obtainable in unit gravity is obtainedwhen the fluid medium, vessel wall, and internal vessel components arecaused to rotate at the same angular rates. The absolute angularrotation rate is chosen to produce the desired orbital path of cells,tissues, and substrates through the fluid medium. This path is primarilydetermined by the sedimentation properties of these suspended particles(through the culture medium), the external gravitational field strength(and direction), angular rotational rates, and vessel dimensions. Theseparameters are related by the appended fluid dynamic calculations thatmay be utilized to choose optimal rotation rates and vessel dimensionsfor specific cultured tissues and culture fluid mediums of variousviscosities. In a specific process, the fluid-culture medium is causedto rotate about a horizontal (or nearly horizontal) axis with respect tothe external gravity vector at an angular rate which optimizes theorbital path of cells, tissues, and attachment substrates suspendedwithin this culture medium so as to optimally control collisionfrequencies, collision intensities, and localization of these particleswith themselves and with the limiting boundaries of the culture vessel.The vessel walls are set in motion relative to the fluid medium andinternal vessel components so as to introduce initially essentially nofluid shear field; however, when appropriate to produce the desiredmulticellular assemblies or for the specific research purpose, such asstudying the effects of shear on the 3-dimensional tissue, a shear fieldmay be introduced and controlled.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one apparatus which may be utilized inthe present invention;

FIG. 2 schematically illustrates the apparatus in FIG. 1 in verticalcross section;

FIG. 3 is a view in cross section along line 3-3 of FIG. 2; and

FIG. 4 is a view in vertical cross section similar to FIG. 2 butillustrating more detail.

FIG. 4A a cross section as in FIG. 4 showing the addition of a timevarying electromagnetic field coil.

FIG. 5 is the orbital path of a typical assembled tissue particle in thenon-rotating reference frame.

FIG. 6 is a graph of the magnitude of deviation of an assembled tissueparticle per revolution.

FIG. 7 is a representative particle path as observed in the rotatingreference frame of the rotating fluid media.

FIG. 8 is a side view of a time varying electromagnetic field coil thatcan house the entire bioreactor to provide a time varyingelectromagnetic field within the bioreactor culture chamber.

FIG. 9 is an end view of the apparatus shown in FIG. 9.

FIG. 10 is an end view of the apparatus shown in FIG. 8 showing thebioreactor therein.

DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention optimizes a group of cultureenvironmental conditions, even as the culturing progresses, in a mannerthat greatly enhances the capability to form and maintain 3-dimensionalliving tissues. The 3-dimensional living tissues are formed andmaintained from cells, which is used herein to include cells in anyform, for example, individual cells or tissue or dissociated sourcecells or cells pre-attached to substrates or intact tissue resections.The process simultaneously minimizes the fluid shear stress, provides3-dimensional freedom for cell and substrate spatial orientation, andincreases localization of cells, tissues, and substrates in a similarspatial region for significant periods during the cell culture. Theprocess of the present invention provides these three criteria that arehereinafter referred to as “the three criteria above”. Transientdisruptions of the stabilized environment are permitted and aretolerated for logistical purposes during initial system priming, sampleacquisition, system maintenance, and culture termination. The cultured3-dimensional tissues (and substrates) increase in size as the cultureprogresses necessitating appropriate adjustment of rotational rates. Theextent or amount of adjustment, depending on vessel dimensions, externalgravitational strength, and absolute sedimentation rate of particlesthrough the media is made in order to optimize these particle paths.Increased external gravity field strength and increased tissue (andsubstrate) sedimentation rate induce restrictions on the choice ofparameters, particularly rotational rate, which ultimately limits theuseful operating range of the process. These same factors place a lowerlimit on the shear stress obtainable, even within the process operatingrange, due to gravitationally induced drift of the particles through theculture media. Calculations and measurements place this minimum shearstress very nearly to that resulting from the particles terminalsedimentation velocity (through the culture media) for the externalgravity field strength. Centrifugal and coriolis induced motion[classical angular kinematics provide the following equation relatingthe Coriolis force to an object's mass (m), its velocity in a rotatingframe (v_(r)) and the angular velocity of the rotating frame ofreference (□): F_(Coriolis)=−2 m (w×v_(r))] along with secondary effectsdue to particle and fluid interactions act to further degrade the shearstress level as the growing tissue increases in size. The process of thepresent invention thus provides an optimization for culturing cells, and3-dimensionality for spatial orientation of cells, substrates, andtissues. The process further provides for selecting vessel dimensionsand rotational rates which minimize collisions of the cultured particleswith vessel walls or internal components. Again, increased gravity andparticle sedimentation rate (which is proportional to size in all casesin our experience) cause restrictions to the process operating range interms of maintaining 3-dimensional freedom.

The particular paths which result from rotation of cells, tissues, andsubstrates about an axis nearly perpendicular to gravity results inthese particles, which may differ greatly in sedimentation rates, toremain spatially localized in similar regions for extended periods oftime. Those time periods may vary from seconds (when sedimentationcharacteristics are large) to hours (when sedimentation differences aresmall). In the process of the present invention this time is extended ascompared to conventional techniques. This allows these particlessufficient time to interact as necessary to form multi-cellularstructures and to associate with each other. The ability to retain thisspatial approximation of particles, which may differ in sedimentationproperties, is degraded by increasing gravity and by increasing particlesedimentation rate. In all of the “three criteria above”; minimizingfluid shear stress, maintaining 3-dimensional freedom for spatialorientation, and maximizing spatial approximation of particles withdiffering sedimentation properties, a degradation of performance isobserved with increasing external gravitational field strength and withincreasing particle sedimentation rate. The process of the presentinvention provides these three criteria above in a manner heretofore notobtained and optimizes the culture environment. It is therefore clearthat further enhancement of this process is obtained in a reducedgravity environment.

This process provides the necessary means for support of respiratory gasexchange, supply of nutrients, and removal of metabolic waste products.This is accomplished either by perfusion of media through an externalmedia perfusion loop, direct injection to the culture media, or exchangeof these molecules across a diffusion membrane. The atmosphericenvironment outside the vessel or circulation loop is controlled withrespect to temperature, humidity, and gas mixture. This is accomplishedby placing the culture and apparatus within a tissue culture incubator.

This cell and tissue culture process provides a stabilized environmentinto which cells or tissues may be introduced, suspended, assembled,grown, and maintained with improved retention of delicate 3-dimensionalstructural integrity. The fluid culture media is initially stabilizedinto near solid body horizontal rotation within the confines of asimilarly rotating vessel wall. The slowest RPM is chosen which will besufficient to produce acceptable particles orbits (with respect to thethree criteria above) upon addition of the initial priming load ofcells, tissue, and substrates. The initial culture may be primed withmetabolically living matter which for example may be separated cells andsubstrates, cells already attached to substrates, or resections oftissue from donor sources.

The addition or priming of particles, cells or substrates, may besimultaneously or sequentially introduced. In most cases the initialparticles with which the culture is primed sediment at a slow rate under0.1 centimeter per second. It is therefore possible, at this earlyculture stage, to select from a broad range of rotational rates(typically 5 to 120 RPM) and vessel diameters (typically 0.5 to 36inches). The slowest rotational rate is advantageous because itminimizes equipment wear and other logistics associated with handling ofthe culture. A vessel diameter is chosen which has the appropriatevolume for the intended quantity of cultured material and which willallow a sufficient seeding density of cells, tissues, and substrates forthe availability of these components. The outward particle drift due tocentrifugal force is exaggerated at higher vessel radii and for rapidlysedimenting particles. This limits the maximum radius of the rotationalculture as a function of the sedimentation properties of the tissuesanticipated in the final culture stages (when large tissues with highrates of sedimentation have formed). Selected levels of shear stress maybe introduced to the culture environment by differential rotation ofvessel components. This is useful to control the rate and size of tissueformation in order to maintain the particle sizes (and associatedsedimentation rate) within a range for which the process is able tosatisfy the three criteria above.

The presence of particles within the stabilized fluid media causessecondary effects to this fluid. These particles move through the fluidmedia under the influence of gravity, centrifugal, and coriolus forces.This motion induces secondary motions to the fluid media. The“stabilized fluid media” referred to in this process is that fluidcondition, particularly the fluid velocity gradients, prior tointroduction of the particles, but which will support a nearly uniformsuspension of these particles upon their introduction. Therefore, thesignificant motion of the fluid media with respect to vessel walls,significant fluid shear stress, and other fluid motions are the resultof the presence of these particles within the fluid media. One exceptionis the case where a fluid velocity gradient and resulting fluid shearstress is intentionally placed on the stabilized fluid for the purposeof limiting the rate or ultimate size of tissue assembly, or for shearstress research purposes.

The path of the particles has been analytically calculated in terms ofthe physical variables important to this tissue culture process. Thesevariables are the vessel dimensions, fluid rotational rate, externalgravitational field strength, and particle sedimentation rate. Theanalysis incorporates the particle motion resulting from gravity,centrifugation, and coriolus effects. A computer simulation of thesegoverning equations allows the operator to model the process and selectparameters acceptable (or optimal) for the particular planned tissueculture. FIG. 5 shows the typical shape of the particle orbit asobserved from the external (non-rotating) reference frame. FIG. 5 is agraph of the radial deviation of a particle from the ideal circularstreamline plotted as a function of RPM (for a typical particlesedimenting at 0.5 cm per second terminal velocity). This graph (FIG. 6)shows the decreasing amplitude of the sinusoidally varying radialparticle deviation as induced by gravitational sedimentation. FIG. 6also shows increasing radial particle deviation (per revolution) due tocentrifugation as RPM is increased. These opposing constraintsnecessitate carefully choosing the optimal RPM to minimize particleimpacts with, or accumulation at the vessel walls. A family of curves isgenerated which is increasingly restrictive, in terms of workable RPMselections, as the external gravity field strength is increased or theparticle sedimentation rate is increased. This family of curves, orpreferably the computer model which solves these governing orbitequations, is utilized to select the optimal RPM and vessel dimensionsfor culture of particles of a given sedimentation rate in a givenexternal gravity field strength. As a typical culture progresses thetissues increase in size and sedimentation rate therefore necessitatingfrequent re-selection of the optimal rotation rate.

A gross determination of corrective rotational rate adjustments inresponse to visual observations may be made. If the particles areobserved to excessively distort inwards on the downward side andoutwards on the upwards side then RPM must be increased. If theparticles are observed to centrifugate excessively to the outer wallsthen the RPM must be reduced. As the operating limits are reached interms of high particle sedimentation rates or high gravity strengths theoperator is unable to satisfy both of these conditions and is forced toaccept degradation in performance as measured against the three criteriaabove. If the particle orbit, FIG. 5, is observed from the rotatingreference frame of the fluid media it is seen to move in nearly circularpath under the influence of the now rotating gravity vector (FIG. 7).The two pseudo forces, coriolis and centrifugal, result from therotating (accelerated) reference frame and cause distortion of theotherwise nearly circular path. Higher gravity levels and higherparticle sedimentation rates produce larger radius circular paths whichcorrespond to larger trajectory deviations from the ideal circular orbitas seen in the non rotating reference frame. In the rotating referenceframe it is now apparent that particles of differing sedimentation rateswill remain spatially localized near each other for long periods of timewith greatly reduced net cumulative separation than if the gravityvector were not rotated. The particles are sedimenting. But they aresedimenting in a small circle (as observed in the rotating referenceframe). This allows particles of differing sedimentation propertiessufficient time to interact mechanically and through soluble chemicalsignals. The sedimentation rates of cells, tissues, and substrates forwhich this process produces significant advantages is from essentiallyzero up to 10 cm per second. As the external gravity field is reduced itis possible to obtain much denser and larger 3-dimensional structures.In order to obtain the minimal shear stress level it is important thatthe vessel walls and internal components be rotated at as nearly aspossible the same rate as the culture media. This minimizes the fluidvelocity gradient induced upon the stabilized fluid media. Fresh orrecycled culture media may be moved within the vessel at a ratesufficient to support metabolic gas exchange, nutrient delivery, andmetabolic waste product removal. This also will slightly degrade theotherwise quiescent culture environment. In the typical case (a tissuewhich sediments at 0.5 cm per second) in which 5 ml per minute isperfused through a 500 ml culture vessel an average flow-speed of 0.001cm per second results. This is quite acceptable and is far slower thaneither gravitationally or centrifugally induced particle motion. Thisperfusion rate may be increased as the tissues' metabolic demandincreases and a large margin is available before significant fluid shearstress results from this fluid perfusion motion. It is necessary tointroduce a mechanism for the support of respiratory gas exchangenutrient delivery, factor delivery, and metabolic waste product removalin order to provide a long term stable culture environment able tosupport significant metabolic loads for periods of hours to months.

It is critical that the culture medium be subjected to a time varyingelectromagnetic force of from 0.05 gauss to 0.5 gauss during the cellgrowth. The time varying electromagnetic field is applied to enhancetissue growth of the cells inside the rotating wall vessel. Theelectromagnetic field may be generated by means such as by directing thecurrent wave form through conducting material wrapped around the outsideof the rotating wall vessel. The field generation system may either berotating with the vessel or fixed, and spaced from, the rotating vessel.

The time varying electromagnetic field is produced by a varyingelectrical potential in the form of a square wave having a frequency ofapproximately 10 cycles per second. Preferably, a current of about 10mille-amps, conducted between the wrappings of the conductive material,produces a stimulatory time varying electromagnetic field extendingseveral centimeters from the conductive material. Typically, the rangeof frequency and oscillating electromagnetic field strength is aparameter, which may be selected for achieving the desired stimulationof particular tissues, cells or genes, and for providing the appropriateamount of up/down regulation of these genes.

In addition to the qualitatively unique tissues that are produced by theprocess of the present invention an increased efficiency with respect toutilization of the total vessel volume for cell and tissue culture isobtained due to the nearly uniform homogeneous suspension achieved. Thisallows an increased number of cells or total tissue mass in the samevolume reactor with less human resources. Many cell type may be utilizedin this process including mammalian, reptile, fish, yeast, and bacteria.The advantage over conventional methods is dependent upon the degree towhich 3-dimensional spatial freedom for metabolically active cellinteractions, assembly, growth, and maintenance is desired to occur in alow (or controlled) shear rate fluid field. The greatest anticipatedadvantage is for mammalian cells that are highly shear sensitive anddepend greatly on multi-cellular architecture and attachment substratesfor their differentiation and function. Fundamental cell and tissuebiology research as well as clinical applications requiring accurate invitro models of in vivo cell behavior are applications for which thisprocess provides an enhancement.

In order to form or to maintain 3-dimensional living tissues from cellsor tissue obtained from natural sources this process provides astabilized quiescent minimal fluid shear stress environment into whichcell attachment substrates, structural substrates, and living cells ortissues may be introduced, suspended, assembled, grown, and maintainedwith retention of delicate 3-dimensional structural integrity.Controlling these particle orbits, particle spatial localizations, andbaseline fluid shear rates, by rotation of the culture environment aboutan axis nearly perpendicular to the gravity vector, produces a processthat optimally counteracts the detrimental sedimentation effects, causedby gravity. These effects limit the ability of conventional techniquesto simultaneously provide a low fluid shear stress environment, provide3-dimensional freedom for tissue assembly and maintenance, and tomaintain a spatial proximity of particles with differing sedimentationproperties.

The process of the present method may be carried out in a number ofspecific apparatus. One such bioreactor is that described in U.S. Pat.No. 5,155,034 with the bioreactor having a coil wound around it or acoil sleeve placed around it to provide the time varying electromagneticforce.

Referring now to FIG. 1, in the overall system illustrated, a main fluidflow loop 10 for growing mammalian cells includes a rotating cellculture reactor vessel 11, and oxygenator 13, a main pump 15 and asupply manifold 17 for the selective input of nutrients, acids, bases,or buffers, such as, sodium hydroxide or fresh medium. The main pump 15provides fresh fluid medium to the oxygenator 13 where the fluid mediumis oxygenated and passed through the cell culture reactor vessel 11. Thereturn spent fluid medium from the cell culture reactor is returned tothe manifold 17 where it receives a fresh charge of nutrients, sodiumhydroxide or liquid medium, as necessary, before recycling by the pump15 through the oxygenator 13 and to the cell culture vessel 11. Thus, acontinuous loop system is provided for the cell growth in the cellculture reactor vessel.

In the system 10, the culture fluid medium is circulated through theliving cell culture in the vessel 11 and around an external life supportloop, as shown in the FIG. 1. In this external loop, adjustments aremade in response to chemical sensors (not shown) that maintain constantconditions within the cell culture vessel 11. Controlling carbon dioxidepressures and introducing acids or bases correct pH. Oxygen, nitrogen,and carbon dioxide are dissolved in a gas exchange system (not shown) inorder to support cell respiration. The closed loop adds oxygen andremoves carbon dioxide from a circulating gas capacitance. In this waythe minimum amount of stored gases may be taken into space if the deviceutilized on a space station or other space vehicles.

FIGS. 2 and 3 schematically illustrate the general details of arotatable bioreactor or cell culture reactor vessel 11 illustrating thepresent invention. In FIGS. 2 and 3, an outer tubular housing 20 isrotatably supported for rotation about a horizontal central axis 21 andabout an input shaft 23 and an output shaft 25 which are aligned withthe central axis. The outer tubular housing 20 has a cylindricallyshaped interior wall 27 and transverse end walls 28, 29 that generallydefine a cylindrically shaped, elongated cell culture chamber 30. A spurgear 32 is attached to one end of the housing 20 and is driven by amotor 33 to rotate the housing about its central horizontal axis 21.

Coaxially disposed about the central axis 21 is tubular inner filterassembly or member 35 that is rotatably mounted on the input shaft 23and is coupled (as shown by the dashed line 36) to the output shaft 25.The output shaft 25, in turn, is rotatably supported, in a stationaryhousing 40 and the output shaft has an externally located spur gear 41which is connected to a drive means 42 for rotating the output shaft 25and the inner filter assembly 35 independently of the outer housing 20.The annular space 30 between the inner filter assembly 35 and theinterior wall 27 of the outer housing member 20 define the annular cellculture chamber 30 located about the horizontal axis 21. Intermediate ofthe outer wall 43 of inner filter assembly 35 and the inner wall 27 ofthe outer member 20 is a blade member system 50 which includes twolengthwise extending blade members 50 a and 50 b which are equiangularlyspaced from one another about the central axis 21. Each of the blademembers 50 a and 50 b at one longitudinal end have a radial arm 52 whichis rotatably supported on the output shaft 25 and at an oppositelongitudinal end 54 have a radial arm 55 which is coupled to the inputshaft 23 (shown by dashed line 56). The input shaft 23, in turn, isrotatably mounted in a stationary housing 60 and the input shaft has aspur gear 61 that is driven by an independent drive mechanism 62 forrotation of the blade members 50 independent of the rotation of theouter member 20.

As shown in FIG. 3, the angular rotation of the three sub-assemblies 20,35 and 50, i.e., the inner filter member 35 the outer housing member 20and the intermediate blade member 50, can be at the same angular rateand in the same direction about a horizontal rotational axis and in thesame direction about a horizontal axis so that there is no relativemovement between the three sub-assemblies. This condition of operationobtains a clinostat suspension of microcarrier beads in a fluid mediumwithin the cell culture chamber without turbulence.

The rotation of the filter can be started and stopped which will causethe turbulence on the surface of the filter and keep the surface clean.The blade members or vanes 50 a and 50 b assist cell culture as theygrow to maintain spatial positions in the rotating fluid medium. This isparticularly helpful for higher density culture particles such as bonecells. By rotating the fluid and the outer wall, the velocity gradientat the wall boundary layer is nearly eliminated.

Referring again to FIG. 2, fluid medium containing fresh nutrients andgases is input, as shown by an arrow 65, to a passageway 66 in thestationary housing 60 and connects to a longitudinal passageway 67 inthe input shaft 23 by virtue of a sealed rotative coupling 70. Thepassageway 67 in the input shaft 23 couples to a radial supplypassageway 72 in an end cap of the outer member 20 by virtue of a sealedrotative coupling 75. The radial supply passageway 72, in turn, connectsto space apart radically directed input passages 78, 79 in the outerhousing 20 member where the input passages 78, 79 are located atopposite ends of the cell culture chamber 30. As shown by the arrows,when fluid is input at both ends of the cell culture chamber 30, thefluid moves radially outward toward the inner wall 27 of the outerhousing member and then moves longitudinally in a horizontal directiontoward a midpoint plane generally indicated by a vertically dashed line80 and then moves radially inwardly toward the outer wall 43 of innerfilter assembly 35. Thus the fluid in the chamber 30 has a generallytoroidal type of motion in radial planes on either side of the mid-pointtransverse plane 80 of the outer member 20. The inner filter assembly 35has openings 82 along its length for exit passage of fluid and, whilenot illustrated in FIG. 2, there is a lengthwise extending filter clothlocated across the openings 82 that prevents microcarrier bead membersin the chamber 30 from exiting through the openings 82. Spent fluid inthe cell culture chamber 30 thus is passed to the interior 85 of theinner filter assembly 35 and exits via a passageway 86 in the outputshaft 25 to a rotative coupling output 88 in the stationary housing 40and to a passageway 89 to the return of the loop passageway forrecharging.

Referring now to FIG. 4, a detailed drawing of the bioreactor of thepresent invention in a preferred form is illustrated. The outer housing20 includes left and right hand cylindrically shaped transverse end capmembers 90, 91 which have facing end surfaces 28, 29 arranged to receivean inner cylindrical tubular glass member 93 and an outer tubular glassmember 94. Suitable pressure seals are provided. Between the inner andouter tubular members 93, 94 is an annular wire heater 96 which isutilized for obtaining the proper incubation temperatures for cellgrowth. The wire heater can be used to also supply a time varyingelectric field to the culture chamber or a separate wire coil 144 can beused to supply the time varying electromagnetic field. The left andright hand end cap members 90, 91 have inner curved surfaces adjoiningthe end surfaces 28, 29 for promoting smoother flow of the fluid withinthe chamber 30. The end cap members 90, 91 have central fluid transferjournal members 94, 95 that are rotatably received respectively on aninput shaft and an output shaft. Each housing journal member 94, 95 hasa flange to seat in a recessed counter bore in an end cap member and isattached by a lock washer and ring 97, 98 against longitudinal motionrelative to a shaft. Each journal member 94, 95 has an intermediateannular recess that is connected to longitudinally extending,circumferentially arranged passages. Each annular recess in a journalhousing is coupled by a radially disposed passage in an end cap member90 or 91 to an input coupling 103, 104. Fluid in a radial passage 78 or79 flows through an annular recess and the longitudinal passages in ajournal member 94 or 95 to permit access fluid through a journal memberto each end of the journal where the access is circumferential about ashaft.

If the time varying electromagnetic force is not applied using theannular wire heater 96, it can be applied by various other methods. Forinstance, FIG. 8-10 illustrate a device that includes a support base145, a cylinder coil support 146 supported on the base with a wire coil147 wrapped around it. The entire bioreactor 148 with the culturechamber is then inserted into the time varying electromagnetic forcedevice.

Attached to the end cap members 90 and 91 are tubular bearing housings105, 106 containing ball bearings which relatively support the housingmember 20 on the input and output shafts 23 and 25. The left handbearing housing 105 has an attached sprocket gear 110 for providing arotative drive for the housing member 20 in a rotative direction aboutthe input and output shafts 23, 25 and the central axis 21. The housing105, 106 also has provisions for electrical take out of the heater wire96 and any other sensor.

The inner filter assembly 35 includes inner and outer tubular members115, 116 having perforations or openings along their lengths and end capmembers 117, 118. The inner tubular member 115 is constructed in twopieces with an interlocking centrally located coupling section and eachpiece attached to an end cap 117 or 118. The outer tubular member 116 ismounted between the end caps 117 and 118.

The end cap members 117, 118 are respectively rotatably supported on theinput shaft 23 and the output shaft 25. The inner member 115 isrotatively attached to the output shaft 25 by a pin and an interfittinggroove 120. A polyester cloth 121 with a ten-micron weave is disposedover the outer surface of the outer member 116 and attached to O-ringsat either end. Because the inner member 115 is attached by a couplingpin to a slot in the output drive shaft 25, the output drive shaft 25can rotate the inner member 115. The inner member 115 is coupled by theend caps 117 and 118 that support the outer member 116. The output driveshaft 25 is extended through bearings in a left hand stationary housing40 and is coupled to a sprocket gear 41.

As illustrated, the output shaft 25 has a tubular bore 120 a thatextends from a port or passageway 89 in the stationary housing 40located between seals to the inner member 115 so that a flow of fluidcan be exited from the inner member 115 through the stationary housing40.

Between the end caps 117 and 118 for the inner member 35 and thejournals 94, 95 in the outer member 20, are hubs 125, 126 for the blademembers 50 a and 50 b. The hub 126 on the input shaft 23 is coupled tothe input shaft 23 by a pin 130 so that the hub 126 rotates with theinput shaft 23. Each hub 125, 126 has axially extending passageways forthe transmittal of fluid medium through a hub.

The input shaft 23 extends through bearings in the right hand stationarybearing housing for rotatable support of the input shaft 23. Alongitudinal passageway 67 extends through the input shaft 23 to alocation intermediate of retaining washers and rings that are disposedin an annular recess 132 between the faceplate and the housing. A radialpassageway 72 in the end cap member 91 permits fluid in the recess toexit from the end cap member 91. While not shown, the passageway 72connects through piping and a Y joint to each of the passages 78 and 79.It will be appreciated that there is a substantial symmetry ofconstruction with respect to the vertical plane defined by line 3-3 ofFIG. 3.

A sample port is shown in FIG. 4, where a first bore 132 extending alonga first axis intersects a corner 133 of the chamber 30 and forms arestricted opening 134. The bore 132 has a counter bore and a threadedring at one end to threadedly receive a cylindrical valve member 136.The valve member 136 has a complimentarily formed tip to engage theopening 134 and protrude slightly into the interior of the chamber 30.An O-ring 140 on the valve member 136 provides a seal. A second bore 141along a second axis intersects the bore 132 at a location between theO-ring 140 and the opening 134. An elastomer or plastic stopper 143closes the bore 141 and can be entered with a hypodermic syringe forremoving a sample. To remove a sample, the valve member 136 is backedoff to access the opening 134 and the bore 141. A syringe can then beused to extract a sample and the opening 134 can be reclosed. No outsidecontamination reaches the interior of the vessel.

In operation, fluid is input to the passageway 66 to the shaftpassageway 97 and thence to the end member passageways 78 and 79 via thepassageway 72. When the fluid enters the chamber 30 via the longitudinalpassages in the journals 95, 94 the fluid impinges on an end surface ofthe blade journals 125, 126 and is dispersed radially as well as axiallythrough the passageways in the journals 125, 126. Fluid passing throughthe journals 125, 126 impinges on the end caps 117, 118 and is dispersedradially. The flow of entry fluid is thus radially outward away from thecentral axis 21 and flows in a toroidal fashion from each end to exitthrough the filter 121 and openings in filter assembly 35 to exit viathe passageways 120 and 89. By controlling the rotational speed anddirection of rotation of the assemblies 20, 30 and 35 any desired typeof fluid action can be obtained. Of major importance, however, is thefact that a clinostat operation can be obtained together with acontinuous supply of fresh medium and oxygen.

Protocol

The methods of the current invention process may be generalized asfollows. An appropriate volume rotating wall vessel, such as a 500 mlrotating wall perfused culture. vessel with an internal vane, is chosen.The vessel is first connected to the external media perfusion conduitsincluding the gas exchange membranes, pump, and media sample ports andis then sterilized, preferably with ethylene oxide gas, and washed withsterile phosphate buffered saline (PBS) then watered and aerated. Thevessel is completely filled with the appropriate media for the cells ortissue to be cultured. The controlled environment incubator thatcompletely surrounds the rotating wall vessel and in which it operatesis set for 5% CO.sub.2, 21% oxygen, and 37 degrees Celsius. The mediaperfusion loop is set at a rate adequate to allow timely equilibrationof the liquid media dissolved gases with the external controlledincubator environment. Cell attachment substrates are introduced eithersimultaneously or sequentially with cells into the culture vessel togive an appropriate density, such as 5 mg of beads per of ml of media,of attachment substrate for the anchorage dependent cells. The culturemedia is then rotated about a horizontal axis. The rotation may begin atabout 10 revolutions per minute, the slowest rate which produces amicrocarrier bead orbital trajectory in which the beads do notaccumulate appreciably at the vessel walls either by gravitationalinduced settling or by rotationally induced centrifugation. The vesselwall, mixing vane, and centerline spin filter are also set at the samerotation (10 RPM) in order to provide essentially no relative motion ofthe culture media with respect to the vessel internal surfaces andtherefore producing the minimal fluid velocity gradients and fluid shearstresses in the culture fluid dynamic environment.

It is recognized that in gravity the relative motion of the rotatingfluid without any particles may be reduced to a level which can not beeasily preserved; however, that when particles are placed into therotating fluid that a measurable or visual change occurs and somerelative motion occurs. However, the shear stress field which results isthe minimum which has been found in a bio-reactor for culturing cells ortissue and therefore, permits the creation of 3-dimensional tissue whichhas not heretofore been possible. This mixture of freely suspendedattachment substrates within the rotating fluid media and rotatingvessel components is allowed to equilibrate for a short period of time,a time sufficient for transient flows to dampen out. The media perfusionrate is set to zero during the cell loading and initial attachment(cells attaching to the microcarrier substrates) phase so as to retainthe small cells within the vessel proper and not to draw them throughthe spin filter and external media perfusion loop where severe celldamage would occur. This initial loading and attachment phase may take 2to 4 hours. The absence of perfusion induced mixing, nutrient delivery,waste product removal, and respiratory gas exchange during this periodis well tolerated due to the small total amount of initial cellularmetabolism and the brevity of this condition. The desired cells aregently injected into the rotating culture media over a short period oftime, (2 minutes) so as to minimize cell damage while passing throughthe syringe and injection port. After injection of the cells iscomplete, the vessel outer wall is quickly (within 1 minute) returned toinitial rotation (10 RPM) to match the angular rotational rate of therest of the system and thereby returning the fluid shear stress field tothe minimal level obtainable for these freely suspended cells andattachment substrates.

All free gas bubbles are then purged via a venting port to assureminimal disruption to the rotating fluid/substrate/cell mixture whichwould result from buoyant gas interfering with the near solid bodyrotating mixture. The cells and beads remain in nearly the same localregion due to the similarity of their orbits and do not achieve a largenet separation from each other. They are allowed to interact with3-dimensional freedom for spatial orientation within the rotating fluidand are not exposed to disruptive shear stresses which would cause celldamage and limit the assembly of delicate 3-dimensional structures.

After the initial loading and attachment phase (2 to 4 hours) theperfusion loop is set at a low flow speed (4.5 ml. per minute) that doesnot interfere with the initial 3-dimensional assembly process. As theculture progresses the size and sedimentation rate of the assembledtissue increases, the system rotational rates must be increased(increasing in increments of about 1 to 2 RPM from about 10 to about 20RPM or more) in order to reduce the gravitationally induced orbitaldistortion (from the ideal circular streamlines) of the now increased (2millimeter) diameter tissue pieces. The rotational rate may be increaseduntil centrifugation is observed to cause accumulation of the tissuepieces at the outer vessel walls that becomes detrimental to further3-dimensional growth of delicate structure. The rapid cell growth andincreasing total metabolic demand necessitates additional intermittentinjections of nutrients. This injection is increased as necessary tomaintain glucose and other nutrient levels. The culture may be allowedto progress beyond the point at which it is possible to select excellentparticle orbits. At this point gravity has introduced constraints whichsomewhat degrades performance in terms of fully 3-dimensional low sheartissue culture.

Every 15 minutes during the total culture period the spin filter isstopped and started at 15-second intervals for 1 minute in order toclear particles from the spin filter surface. This prevents accumulationof substrates, cells, and debris on the filter. Samples of the growingtissue may be withdrawn as desired from a syringe attached to the sampleport. The vessel outer wall may be temporarily stopped to allowpractical handling of this syringe connected to the vessel.

EXAMPLE I

Preparation

A 3-dimensional rat bone cell culture was produced in the followingmanner. The bio-reactor, slow turning lateral vessel (STLV) was preparedby washing detergent and germicidal disinfectant solution (Roccal II) atthe recommended concentration for disinfection and cleaning followed bycopious rinsing and soaking with high quality deionized water. Thebioreactor was sterilized by autoclaving then rinsed once with culturemedium.

Inoculation

The 100 ml. volume STLV was filled with cell culture medium consistingof minimum essential medium (MEM) with Earle's salts, growthsupplements, antibiotics and 10% fetal bovine serum. After equilibrationfor one hour in the CO.sub.2 incubator, the substrate consisting ofcollagen coated dextian polymer, Cytodex 3 microcarrier beads (PharmaciaFine Chemicals, Uppsala, Sweden) were suspended in a small volume ofculture medium and loaded into the device. An empty syringe attached toone of the sampling ports functioned as a compliant volume reservoirduring inoculation to receive the displaced media. The final beadconcentration was 5 mg./ml. of vessel volume.

A volume of culture medium was injected to completely fill the device.All air bubbles were removed using a syringe attached to a sample porton the vessel. The device containing suspended beads was placed in a 5%CO.sub.2 environment at 37.degree.C. to equilibrate for about 30minutes.

Mono-dispersed primary rat osteoblast calvarium bone cells weresuspended in a small volume of cell culture medium and inoculated intothe vessel by injection through a sample port. The cell seeding densitywas approximately 10 cells per bead. A second syringe was attached toanother port and served as a compliant volume reservoir for the vessel.The device was placed on the rotation base and connected to the airpump. The motor was switched on and the rotation rate was set atapproximately 16 RPM.

Cell Attachment and Three Dimensional Growth

At 24 hours, microscopic observation showed that the cells were wellattached and flattened on the surface of the beads. The beads and cellswere not associated into higher order structures at this point. Verylittle orbital path distortion or centrifugation was observed. Themedium was changed to remove non-viable floating cells. On day three,the medium was changed again to assure nutrient supply to the cells. Atday four cells were in good condition. To replenish nutrients yet retaingrowth factors secreted by the cells, three-fourths of the medium wasremoved and the same volume of fresh medium was added. The sameprocedure was repeated on day 5.

On day 5, microscopic observation of samples showed rounded assembliesof beads that were larger than seen on the previous day. Also noted werecells spanning spaces between beads. The airflow into the device wasturned up to provide more oxygen to the rapidly growing culture. At thispoint the assemblies were observed to fall radially inwards on the“down” side and outwards on the “upside”.

On the 6th day the culture was transferred to a larger 250 ml volumeSTLV device. On day 8, ordered assemblies of beads were very large (1-2mm) with 8 to 15 beads in the assemblies and more three-dimensionalstructure was noted. The medium was changed at this time. At this pointsome accumulation of the tissue assemblies was occurring at the outervessel wall. This centrifugation effect was quite gentle.

The cells were maintained in the three dimensional structure for 17 dayswith additional medium changes on days 9, 10 and 12 and additions ofglucose on days 11, 15 and 16. There was no evidence of mechanicaldamage and the size of the cell/bead assemblies did not exceed theability of the device to suspend the particles. The assemblies were of 1to 2 mm in size and consisted of cells of mixed morphology which may bethe indicia of beginning differentiation. The run was terminated bychoice of the investigators.

EXAMPLE 2

Formation of “artificial tissue” in free suspension

Preparation:

The rotating reactor vessel system (RWPV) consisting of a 500 ml cellculture vessel, a hollow fiber oxygenator, a prototype diaphragm pump,an in-line pH sensor, sample ports and a peristaltic pump for infusionof fresh medium were assembled, sterilized by ethylene oxide (ETO) andaerated for two days. The vessel was then loaded with phosphate bufferedsaline (PBS) to rinse and remove residual ETO. During this step, a leakwas discovered in the oxygenator and unit was replaced using steriletechniques. The system was then loaded with culture medium and placed inthe CO.sub.2 incubator. After remaining sterile for at least two days,the RWPV system was loaded with cells and substratum as described below.

Inoculation

Cytodex 3 microcarrier beads (Pharmacia Fine Chemicals, Uppsala, Sweden)were reconstituted according to standard laboratory procedures,suspended in Microcarrier Medium (MM), containing 20% fetal calf serum,100 units of penicillin/ml and 100 ug of streptomycin/ml, loaded into a50 ml syringe and injected into the vessel. The bead density in thevessel was 5 mg/ml of vessel volume.

The system was then loaded with Baby Hamster Kidney (BBK21) cells atpassage level 56. To achieve this, ampules of frozen stock BHK21 cellswere thawed. The cells were suspended in 50 ml of growth medium (MM) andput into a 20 ml syringe for injection into the culture vessel. Thefinal cell seeding density was approximately 6 cells/bead. Cells wereloaded into the vessel at 9:30 AM.

The system parameters were as follows. The constant volume diaphragmpump was timed to circulate the medium at 4.5 ml/min. The pump rate wasturned up to 20 mi/min four days after addition of cells and beads inorder to increase the oxygen delivery to the reactor vessel. The pumpsystem delivered 0.7 ml every two seconds. The rotation rate of thevessel and the mixing blades were set at 15 to 20 RPM and remained thereduring the test. The spin filter rotation rate was 25 to 30 RPM. Thisproduced a very low turbulence environment resulting in a cell/beadsuspension upon introduction of those particles.

Cell Attachment and Three Dimensional Growth:

In order to assess the rate of attachment of cells to the substratebeads, samples of the cell-bead suspension were removed for cell countsand microscopic observation at 2, 4 and 6 hours after loading the vesselwith cells. Many of the cells attached to the beads within two hours andflattened on the surface of the beads which is an essential statepreliminary to growth of the cells. Early in this test microscopicobservation showed that some of the mono-dispersed cells clumped ingroups of 10 to 30 cells. There did not appear to be any mechanicaldamage or damage due to cells striking the vessel wall. At 4 hours,these clumps of cells had attached to beads and were flattened on thesurfaces. No orbital distortion or centrifugation effects were visibleat this point.

At 24 hours some beads were covered with cells but there were also manyfloating cells. At this time, fresh medium was perfused into the vessel.Four hours later, almost all of the cells had attached to beads andthere was no evidence of cell-cell aggregates as seen earlier. The poorappearance at 24 hours was probably due to toxicity from the newoxygenator used to replace the original one that leaked during set up.

At 48 hours the cell growth rate began to increase rapidly. Glucose,glutamine (nutrients) and sodium hydroxide for pH control were added onday four to compensate for cell metabolism and depletion of nutrients.

At five days, aggregates of cells and beads were noted and the mediumwas changed to a low percent serum (2%) medium to slow cell metabolismand growth. The cells continued to grow, however, and reached a maximumdensity of 148 cells/bead at day 7. Very large and uniformed assembliesof about 1 to 2 mm formed and did not disaggregate during the remainderof the test. Ordered uniform assemblies of 8 to 10 beads formed in 3dimensional arrays over which the cells grew into a smooth membrane-likeconfiguration of elongated fibroblastoid morphology. Multiple layers ofthese cells were apparent by microscope examination both on the surfaceand in the inter-bead spaces. The cells were placed in ordered layerswith cell membranes immediately adjacent to each other. The test wasterminated at 10 days. The size of most of the tissue-like aggregates ofcells on beads did not exceed the limits of the rotating wall vanedvessel to freely suspend the aggregates in a quiescent 3-dimensionalenvironment, unrestricted by internal boundaries. Some very largeassemblies, 3 to 8 mm, were observed. These rapidly sedimenting tissueswere observed to exhibit grossly distorted orbital paths andcentrifugation to the outer vessel walls. High-energy impacts andvigorous “rolling” effects were observed. These were considered beyondthe process capacity to retain the quiescent, low shear, 3-dimensionalculture environment.

Having fully described this new and unique invention,

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. In a method for culturing mammalian cells to produce invitro multi-cellular, three-dimensional assemblies of predominatelyliving mammalian cells with predominately adjacent cell membranesevidencing minimal mechanical damage, said method including the stepsof: providing a bioreactor having a culture chamber rotatable about anapproximately horizontal longitudinal axis, means to controllably rotatesaid culture chamber, means to introduce an oxygen-containing fluidthroughout said culture chamber and means to remove metabolic wasteproducts therefrom; filling said culture chamber completely with a fluidnutrient media, cell attachment substrates, and mammalian cells of asingle type, to establish a culturing environment; rotating said culturechamber about its longitudinal axis while introducing anoxygen-containing fluid to said culture chamber and removing metabolicwaste products therefrom to maintain said culturing environment;controlling the rotation of said culture chamber such that the fluidmedium except during transient periods as the following simultaneousproperties: a. localization of cells and attachment substrates withsimilar or differing sedimentation properties in a similar spatialregion, b. freedom for three dimensional spatial orientation ofassemblies formed by the culturing of the cells, c. low shear andessentially no relative motion of said culturing environment withrespect to the boundaries of the chamber; and determining the trajectoryof the assemblies, in response to said determining step: increasing thespeed of rotation of the culture chamber if the assemblies fallexcessively inward and downward on the downward side of the rotationcycle and excessively outward and insufficiently upward on the upgoingside of the rotation cycle to prevent wall impact; or decreasing thespeed of rotation of the culture chamber in response to excessiveaccumulation of the assemblies near the outer wall of the culture vesselso as not to restrict three dimensional assembly, and as the size of theassemblies increase beyond the capability to fully satisfy the abovethree properties by further increase of the speed of rotation, selectinga rotational rate to visually obtain minimal collision frequency andintensity, the improvement comprising: subjecting the contents of theculture chamber to a time varying electromagnetic force of from about0.05 to 0.5 gauss while culturing the cells in the chamber.
 8. In amethod for growing mammalian cell cultures comprising the step ofproviding a bioreactor having a culture chamber formed of a coaxialtubular housing, intermediate circumferentially disposed blades and acentral tubular filter about a common horizontal axis of rotation eachbeing independently rotatable of one another; disposing a fluid nutrientmedium having a first density, mammalian cells, and discrete suspensionmaterials having a different density from the density of the fluidnutrient medium in said culture chamber; controlling the rotation ofsaid culture chamber and thereby the fluid nutrient medium for placingthe mammalian cells and the discrete suspension materials in suspensionat discrete spatial locations in the fluid nutrient medium and out of aninterference relationship with one another by virtue of the rotation;introducing a controlled input of fresh fluid nutrient medium to saidculture chamber and removing a controlled output of fluid nutrientmedium from said culture chamber while said fluid nutrient medium insaid culture chamber is rotated about said axis of rotation, theimprovement comprising: subjecting the contents of the culture chamberto a time varying electromagnetic force of from about 0.05 to 0.5 gausswhile culturing the cells in the chamber.
 9. The method set forth inclaim 8 wherein the rotation of the central tubular filter isintermittently at a different rotational speed than the tubular housingto create a transient shear stress thereby clearing the surface of thecentral tubular filter and preventing clogging.
 10. The method set forthin claim 8 wherein the rotation of the circumferentially disposed bladesis a different rotational speed than the tubular housing to assist thediscrete suspension materials and the mammalian cells to maintain theirspatial locations.
 11. The method set forth in claim 8 wherein, when therotation of the tubular housing is stopped, the circumferentiallydisposed blades may be rotated to maintain the discrete suspensionmaterials and the mammalian cells in suspension.
 12. (canceled) 13.(canceled)
 14. In a method for growing mammalian cell culturescomprising the steps of providing a bioreactor having a housingcompletely filled with a fluid nutrient medium with a first density,mammalian cells, and discrete suspension materials having a differentdensity from the density of the fluid nutrient medium and rotating thehousing about a rotational axis; controlling the rotation of the fluidnutrient medium for placing the discrete suspension material andmammalian cells in suspension at discrete spatial locations in the fluidnutrient medium and out of an interference relationship with one anotherby virtue of the rotation of the fluid nutrient medium and axis ofrotation; while rotating the housing containing the fluid nutrientmedium, radially introducing fresh fluid nutrient medium into thehousing at a second input location where said first input location andsaid second input location are at opposite ends of the housing andlocated adjacent the rotational axis, while removing fluid nutrientmedium from a location intermediate of said first and said second inputlocation so that the flow of fluid medium is toroidal from each end ofthe housing, the improvement comprising: subjecting the contents of thehousing to a time varying electromagnetic force of from about 0.05 to0.5 gauss while culturing the cells in the housing.
 15. The method asset forth in claim 14 and further including the step of: maintaining theinput and output volume of fluid nutrient medium such that there is nohead space in the housing.
 16. The method as set forth in claim 14wherein the rotation of the housing is at a rate of rotation to minimizethe velocity gradient between the inner wall of the housing and thefluid nutrient medium.
 17. The method as set forth in claim 14 andfurther including the step of recirculating the removed fluid medium tosaid first input location and further including the step of introducingconstituent materials to the fluid medium prior to introducing fluidnutrient medium into the housing.
 18. The method set forth in claim 14wherein said bioreactor further includes a central tubular filter andwherein the removal of the fluid nutrient medium is accomplished byintermittently having a different rotational speed of the centraltubular filter than the rotational speed of the tubular housing tocreate a transient shear stress thereby clearing the surface of thecentral tubular filter.
 19. In a method for growing cell culturescomprising the steps of providing a bioreactor formed of a tubularhousing completely filled with a fluid nutrient medium discretesuspension materials, and mammalian cells are disposed in the fluidnutrient medium and rotating said housing about a horizontal rotationalaxis; controlling the rotation of the fluid nutrient medium for placingthe discrete suspension materials and the mammalian cells in suspensionat discrete spatial locations in the fluid nutrient medium and out of aninterference relationship with one another by virtue of the rotation ofthe fluid nutrient medium and horizontal axis of rotation; whilerotating the housing containing the fluid nutrient medium, introducingfresh fluid nutrient medium into the housing at input locations, oneadjacent each end of the housing, while removing fluid nutrient mediumfrom the housing at another location, such input being radiallydischarged relative to the rotational axis whereby fluid turbulenceresulting from the incoming fluid nutrient medium is maintained at aminimum thereby setting up a flow field which is toroidal from each endof the housing, the improvement comprising: subjecting the contents ofthe housing to a time varying electromagnetic force of from about 0.05to 0.5 gauss while culturing the cells in the housing.
 20. The method asset forth in claim 19 wherein, when the rotation of the tubular housingis stopped, the discrete suspension materials and the cells aremaintained in suspension by rotation of a pair of circumferentiallydisposed blades.
 21. (canceled)
 22. A method for culturing mammaliancells in a bioreactor comprising the steps of: a. placing a fluidculture medium and at least one cell in a cell culture reactor vessel toinitiate a cell culture environment; b. rotating the culture reactorvessel via a means for rotating the culture vessel about a horizontalcentral axis at a rotation speed; c. controlling the rotation of theculture reactor vessel by adjusting the rotation speed while at the sametime maintaining the cell culture environment; and d. exposing the cellsto a time varying electromagnetic force from about 0.05 to about 0.5gauss.
 23. The method of claim 22, wherein the cell culture environmenthas the properties of collocation of the fluid culture medium and thecells, essentially no relative motion of the fluid culture medium withrespect to the culture reactor vessel, and freedom for the threedimensional spatial orientation of the cells.
 24. The method of claim22, wherein the cell culture environment is maintained by perfusion. 25.The method of claim 22, wherein the cell culture reactor vessel has aninput means for the introduction of a volume of oxygen bearing fluidculture medium and an output means for the removal of waste bearingfluid culture medium to a waste.
 26. The method of claim 22, wherein thefluid culture medium has a main fluid flow loop comprising a supplymanifold, a pump, an oxygenator, a cell culture reactor vessel, and awaste.
 27. The method of claim 26, wherein the main fluid flow loopenriches the fluid culture medium with at least one selected from thegroup consisting of growth factors, oxygen, nutrients, acids, bases,buffers, and fresh medium prior to entering the cell culture reactorvessel.
 28. The method of claim 26, wherein a volume of the fluidculture medium is removed from the cell culture reactor vessel to awaste.
 29. The method of claim 22 wherein the bioreactor is located inunit gravity.
 30. The method of claim 22 wherein the bioreactor islocated in microgravity.
 31. The method of claim 22, wherein the fluidculture medium comprises a cell attachment substrate.
 32. The method ofclaim 31, wherein the cell attachment substrate is at least onemicrocarrier bead.
 33. The method of claim 22 wherein the rotation speedis from about 5 to about 100 RPM.
 34. The method of claim 22, whereinthe step of placing the cells and fluid culture medium into the cellculture reactor vessel comprises adding the cells to the fluid culturemedium wherein either the cells or the fluid culture medium are alreadypresent in the cell culture reactor vessel.
 35. The method of claim 22,wherein the step of placing the cells and fluid culture medium into thecell culture reactor vessel comprises mixing the cells and fluid culturemedium outside the cell culture vessel and then adding the mixed cellsand the fluid culture medium to the cell culture reactor vessel.
 36. Themethod of claim 22, wherein the method is carried out in unit gravity.37. The method of claim 22 wherein the method is carried out in lessthan unit gravity.